蛇床子素促进人骨肉瘤细胞株SAOS-2凋亡

目的:观察蛇床子素(osthole)对人骨肉瘤细胞SAOS-2增殖和凋亡的影响及潜在的调控机制。方法:采用MTT法、TUNEL染色技术和流式细胞术检测不同浓度蛇床子素对骨肉瘤细胞凋亡的影响;Western blot检测蛇床子素对骨肉瘤细胞中与细胞凋亡密切相关的蛋白(Bax、Bcl-2)的变化。结果:蛇床子素作用于SAOS-2细胞后,MTT结果显示SAOS-2细胞的活力受到明显抑制,且与蛇床子素浓度和时间相关;Western blot结果显示细胞中的促凋亡蛋白Bax表达上调,抗凋亡蛋白Bcl-2表达明显减弱,且呈剂量依赖性。结论:蛇床子素可显著抑制人骨肉瘤细胞的增殖且促进其凋亡的作用,可能与上调凋亡蛋白Bax和下调抗凋亡蛋白Bcl-2的表达有关。     

金雀异黄素对不同淋巴转移能力的小鼠肝癌细胞株的抑制作用

MTT法检测大豆提取物金雀异黄素（genistein,Gen）对不同淋巴转移能力的腹水型小鼠肝癌细胞株HepA-H和HepA-L的生长抑制作用。应用电镜观察细胞形态和流式细胞术检测细胞凋亡。将HepA-H和HepA-L接种NIH小鼠,制备荷瘤动物模型,Gen腹腔给药后第14天取淋巴结观察,并计算转移率。TUNEL法检测淋巴结凋亡细胞,并计算凋亡指数。实验发现：（1）Gen对两株细胞均具有良好的生长抑制作用,且对HepA-H的抑制优于HepA-L;（2）Gen在体外诱导两株细胞发生凋亡,且HepA-H的凋亡率高于HepA-L,并呈量效关系;（3）Gen抑制荷瘤动物肿瘤生长和肿瘤淋巴转移,且对HepA-H的抑制强于HepA-L,实验组H的凋亡指数（3．87％）也显著高于实验组L（1．69％）。故研究认为Gen显著抑制HepA-H和HepA-L肝癌细胞淋巴转移的作用机制,可能与诱导细胞凋亡有关,对高淋巴转移细胞的抑制作用强于低淋巴转移细胞,提示Gen抗肿瘤作用具有一定的选择性。     

丙二醛对体外培养下小鼠骨髓间充质干细胞凋亡的诱导作用

为了探索丙二醛对小鼠骨髓间充质干细胞(MSCs)凋亡的诱导作用及其机制,在不同浓度的丙二醛培养体系中孵育MSCs 24 h,用TUNEL法、流式细胞术检测MSC凋亡率,并用实时定量RT-PCR、Western印迹检测Bcl-2、Bax及Caspase-3基因的表达水平。结果发现,MDA能浓度依赖性地增加TUNEL阳性细胞百分率、亚G1峰细胞百分率,同时下调Bcl-2 mRNA及蛋白的表达,上调Bax mRNA和Caspase-3 mRNA及蛋白的表达.这些结果表明:在体外培养条件下,丙二醛可诱导小鼠骨髓间充质干细胞的凋亡,其作用机制与Bcl-2、Bax和Caspase-3基因表达水平的变化有关。     

AS-mCLB1重组质粒体外抗肿瘤及化疗增敏作用

研究反义全长小鼠细胞周期蛋白B1重组质粒(pAS-mCLB1)体外抗肿瘤及化疗增敏作用。扩增后少量抽提并纯化pAS-mCLB1,通过脂质体将其转染入小鼠露易丝肺癌(LL/2)细胞,RT-PCR和Western印迹测定细胞内细胞周期蛋白B1的表达,观察转染后细胞形态变化,MTT法检测细胞增殖活性,流式细胞仪检测细胞周期及凋亡。细胞转染48h后,用化疗药物健择(gemcitabine;0.2μmol/L)处理24h,MTT法测定健择对细胞的杀伤作用。研究提示pAS-mCLB1转染后LL/2细胞形态明显异常,细胞内细胞周期蛋白B1表达显著下调,细胞周期阻滞于G1期,增殖受抑,凋亡增加;健择对pAS-mCLB1转染后LL/2细胞的杀伤作用显著增强。重组质粒pAS-mCLB1体外具有明显的抗肿瘤作用,并能增强肿瘤细胞对化疗药物的敏感性,估计上述作用与其下调肿瘤细胞内细胞周期蛋白B1表达,从而诱导细胞周期阻滞及凋亡等作用相关。     

紫草素对肝癌细胞SMMC-7721凋亡的影响及其机制

目的: 探讨紫草素对肝癌SMMC-772细胞的作用及分子机制。方法: SMMC-7721细胞分别经0、5、20、80、320 ng/ml的紫草素作用0 h、24 h、48 h和72 h后,适时采用CCK8法观察该细胞增殖的活性,hoechst染色分析细胞的核型变化,流式细胞仪检测细胞凋亡水平,Western blot证实细胞内蛋白表达水平的改变,通过小鼠体内实验观察该药的抑瘤作用。结果: 本研究体外实验发现紫草素可显著抑制SMMC-7721细胞的增殖活性并诱导其凋亡(P＜0.01),上调基因p53的表达,并抑制AKT、PI3K蛋白的磷酸化,体内实验也证实紫草素可显著抑制荷瘤小鼠肿瘤的生长(P＜0.01),作用效果随用药剂量和时间的增加而增加。结论: 紫草素可通过影响PI3K/AKT信号通路抑制SMMC-7721细胞的增殖,且诱导其凋亡,具有潜在的抗肝癌作用。     

芹菜素通过升高ROS促进不同细胞凋亡的实验研究

芹菜素是一种具有抗氧化、抗肿瘤和抗炎活性的天然植物黄酮,本研究应用MTT法测芹菜素对小鼠脾细胞和S180细胞增殖的影响,流式细胞仪测细胞凋亡和细胞内活性氧水平。结果显示:芹菜素剂量依赖地抑制小鼠脾细胞和S180细胞增殖,促进脾细胞和S180细胞凋亡,显著升高脾细胞和S180细胞内活性氧水平。这些结果提示:芹菜素可能通过升高不同细胞内ROS水平,诱导细胞凋亡,抑制细胞增殖,具有进一步研究开发价值。     

蛇床子素对人胶质瘤U251细胞抗增殖作用的研究

目的:研究蛇床子素对人胶质瘤U251细胞的抗增殖作用和可能的机制.方法:不同浓度蛇床子素处理U251细胞后,MTT法检测细胞活力,流式细胞法检测细胞周期和凋亡情况.结果:①蛇床子素显著抑制U251细胞的增殖.②蛇床子素诱导U251细胞发生G2/M期阻滞和凋亡.③蛇床子素处理后的U251细胞PI3K/Akt信号通路活性受到明显抑制.结论:蛇床子素通过抑制PI3K/Akt信号通路抑制人胶质瘤U251细胞的增殖.     

软骨多糖诱导小鼠S180细胞凋亡的作用机制

目的研究软骨多糖对S180荷瘤小鼠的作用,并探讨其抑瘤作用机制。方法采用小鼠肉瘤S180细胞建立动物腹水瘤模型,通过腹腔注射软骨多糖进行治疗,治疗期间抽取腹水瘤细胞进行细胞生物学分析。通过HE染色,流式细胞术、TUNEL法检测细胞形态学方面、细胞周期及凋亡率的变化情况;通过免疫荧光方法检测Fas、增殖细胞核抗原(PCNA)的表达情况。结果软骨多糖可以明显提高S180荷瘤小鼠的生存率,细胞形态学观察可见细胞出现细胞质浓缩、核固缩及凋亡小体等现象。软骨多糖作用后的S180细胞,其细胞周期被阻遏于G2/M期,Fas蛋白的表达水平于给药24 h后升高,增殖细胞核抗原PCNA表达下降。结论软骨多糖可能通过影响肿瘤细胞周期和Fas、PCNA蛋白的表达来诱导S180细胞凋亡,并显著抑制肿瘤细胞的生长,延长S180荷瘤小鼠的生存时间,研究证实动物软骨多糖具有潜在的药用价值。     

下调CDH17基因对那可丁耐药性人胃癌细胞作用的分析

探讨下调肝肠钙黏连蛋白(CDH17)基因对抑制那可丁耐药性人胃癌MGC-803细胞增殖的分子机制,为临床治疗胃癌提供新的治疗手段。本研究以人胃癌MGC-803细胞为研究材料,通过体外MTT实验检测细胞活力、流式细胞术观察细胞增殖与凋亡情况,蛋白免疫印迹检测凋亡通路相关蛋白的表达水平变化。那可丁药物处理实验和MTT实验表明,下调CDH17可以提高人胃癌MGC-803细胞对那可丁的敏感性;流式细胞术观察结果表明,下调CDH17基因促进引起细胞凋亡;蛋白免疫印迹实验表明线粒体途径参与那可丁诱导人胃癌MGC-803细胞凋亡的过程。综上所述,下调CDH17基因通过提高人胃癌MGC-803细胞对那可丁的敏感性,引起线粒体途径相关蛋白功能障碍促进细胞凋亡,从而抑制胃癌细胞的增殖。     

汉黄芩素抗骨肉瘤143B细胞的实验研究

目的:观察汉黄芩素对人骨肉瘤细胞系143B增殖和凋亡的影响,并探讨其可能的作用机制。方法:采用体外培养人成骨肉瘤细胞系143B,CCK-8实验检测不同浓度汉黄芩素对骨肉瘤143B细胞增殖抑制作用;流式细胞术分析汉黄芩素对癌细胞周期分布及凋亡的影响;Western Blot检测凋亡相关蛋白Bax、Bcl-2、cleaved caspase-9和cleaved caspase-3的表达水平。结果:CCK-8结果显示汉黄芩素以时间、浓度依耐性的方式抑制骨肉瘤143B细胞的增殖;流式细胞术结果表明汉黄芩素可导致骨肉瘤细胞周期阻滞于G0/G1期并以浓度依赖的方式诱导骨肉瘤细胞凋亡;Western Blot检测结果证明,汉黄芩素可上调骨肉瘤细胞中促凋亡蛋白Bax、cleaved caspase-9、cleaved caspase-3的表达,而下调抑制凋亡蛋白Bcl-2。结论:汉黄芩素抑制骨肉瘤细胞增殖、导致细胞周期阻滞,促进其凋亡,并呈现时间和浓度依赖性,汉黄芩素激活Caspase凋亡途径及诱导细胞周期阻滞可能是其抗骨肉瘤的作用机制。     

The regulation of ERK and p-ERK expression by cisplatin and sorafenib in gastric cancer cells

Previous studies have reported strong antitumor effects of cisplatin and sorafenib. Our results indicated that cisplatin and sorafenib exhibited anti-tumor effects on gastric cancer cells. They significantly inhibited gastric cell growth and induced apoptosis. They effectively inhibited gastric cancer cell proliferation and induced G0/G1 phase arrest. Western blotting analysis indicated that it also promoted the phosphorylation extracellular signal regulated kinase (p-ERK). Moreover, cisplatin and sorafenib played a synergistic antitumor effect. These results suggested that the antitumor mechanism of cisplatin and sorafenib involved altering the cell cycle and stimulating ERK phosphorylation in the ERK signaling pathway.     

Effect of epiberberine from Coptis chinensis Franch on inhibition of tumor growth in MKN-45 xenograft mice

Background and purposeGastric cancer is one of the major malignancies worldwide. Epiberberine (EPI) is a major alkaloid from Coptis chinensis Franch and the antitumor property of EPI remains poorly understood.MethodThe inhibition on gastric cancer cells was observed by MTT assays and colony formation experiments. The apoptosis, cell cycle, and reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) in gastric cancer cells were analyzed by Flow cytometry. The anti-tumor effect of EPI was evaluated with the MKN-45-beraring nude mice, and the potential mechanisms were explored by RNA-seq, qPCR, siRNA silencing and western blotting.ResultsEPI inhibited the proliferation of human gastric cancer cell lines MKN-45 (harboring wild-type p53) and HGC-27 (harboring mutant p53) in a dose dependent manner. EPI induced the apoptosis and cell cycle arrest in these two cell lines, of which MKN-45 cells are more sensitive to EPI than HGC-27 cells. Further experiments indicated that EPI induced the accumulation of ROS and decreased of ΔΨm in MKN-45 cells. The significant differentially expressed genes obtained by RNA-seq were distinctly enriched in the p53 signaling pathway. The apoptosis induced by EPI in MKN-45 cells would be effectively inhibited with the treatment of p53 siRNA and p53 inhibitor PFT-α. Western blotting demonstrated that EPI diminished the expression of Bcl-2 and XIAP, and increased those of p53, Bax, p21, p27, Cytochrome C and Cleaved-caspase 3. Animal experiments confirmed that EPI significantly alleviated tumor growth in MKN-45 xenograft mice via p53/Bax pathway.ConclusionsThese data indicated that EPI could be a novel anti-tumor candidate against MKN-45-related gastric cancer via targeting p53-dependent mitochondria-associated pathway.     

Artesunate induces apoptosis,autophagy and ferroptosis in diffuse large B cell lymphoma cells by impairing STAT3 signaling

Artesunate (ART), a water-soluble derivative of artemisinin, has been reported to exert antineoplastic effects via diverse mechanisms in various types of cancer. Therefore, understanding the underlying mechanism of action of ART in distinct cancer types is indispensable to optimizing the therapeutic application of ART for different types of cancer. The present study aimed to investigate the cellular and molecular mechanisms responsible for the antineoplastic effects of ART in diffuse large B cell lymphoma (DLBCL) cells. Cell proliferation was measured using Cell Counting Kit-8 and colony formation assays. The levels of apoptosis and cell cycle distribution were investigated using flow cytometry. In addition, western blotting was used to analyze the expression levels of ART-induced apoptosis-, autophagy- and ferroptosis-related proteins. Monodansylcadaverine staining was performed to determine the levels of autophagy. Moreover, malondialdehyde and reactive oxygen species assays were used to determine the levels of ferroptosis. The results of the present study revealed that ART inhibited proliferation, and induced apoptosis, cell cycle arrest, autophagy and ferroptosis in DLBCL cells. Pharmacological inhibition of autophagy and ferroptosis alleviated the increased levels of apoptosis induced by ART. Notably, ART was found to exert its effects via inhibition of STAT3 activation. The genetic knockdown of STAT3 enhanced ART-induced autophagy and ferroptosis, and concomitantly upregulated the expression levels of apoptosis- and cell cycle-related proteins. In conclusion, the findings of the current study suggested that ART may induce apoptosis and cell cycle arrest to inhibit cell proliferation, and regulate autophagy and ferroptosis via impairing the STAT3 signaling pathway in DLBCL cells.     

Antiproliferative mechanism of a cannabinoid agonist by cell cycle arrest in human gastric cancer cells

For gastric cancers, the antineoplastic activity of cannabinoids has been investigated in only a few reports and knowledge regarding the mechanisms involved is limited. We have reported previously that treatment of gastric cancer cells with a cannabinoid agonist significantly decreased cell proliferation and induced apoptosis. Here, we evaluated the effects of cannabinoids on various cellular mediators involved in cell cycle arrest in gastric cancer cells. AGS and MKN-1 cell lines were used as human gastric cancer cells and WIN 55,212-2 as a cannabinoid agonist. Cell cycles were analyzed by flow cytometry and western blotting. Treatment with WIN 55,212-2 arrested the cell cycle in the G0/G1 phase. WIN 55,212-2 also upregulated phospho-ERK1/2, induced Kip1/p27 and Cip1/WAF1/p21 expression, decreased cyclin D1 and cyclin E expression, decreased Cdk 2, Cdk 4, and Cdk 6 expression levels, and decreased phospho-Rb and E2F-1 expression. ERK inhibitor decreased the proportion of G0/G1 phase which was induced by WIN 55,212-2. Inhibition of pAKT led to cell cycle arrest in gastric cancer cells. Cell cycle arrest preceded apoptotic response. Thus, this cannabinoid agonist can reduce gastric cancer cell proliferation via G1 phase cell cycle arrest, which is mediated via activation of the MAPK pathway and inhibition of pAKT.     

Inhibition of two gastric cancer cell lines induced by fucoxanthin involves downregulation of Mcl-1 and STAT3

Fucoxanthin is a natural carotenoid that had never been previously demonstrated to have anti-tumor effect on human gastric adenocarcinoma SGC-7901 or BGC-823 cells. Here it was found to inhibit proliferation and induce apoptosis through JAK/STAT signal pathway in these cells; the mechanism by which this occurred was investigated. We find that fucoxanthin significantly increased the number of apoptotic cells by propidium iodide (PI) dye staining and flow cytometry. Fucoxanthin (50 or 75 μM) induced SGC-7901 cells cycle arrest at S phase, while BGC-823 cells arrest at G2/M phase. RT-PCR and western blot analysis revealed that the expressions of Mcl-1, STAT3 and p-STAT3 were obviously decreased by fucoxanthin in a dose-dependent manner. Synthetic siRNA targeting Mcl-1 was transfected into cells which had no effect on expressions of STAT3. After pretreatment with AG490 (50 μM) which led to blocking of the JAK/STAT signal pathway, the reductive expressions of Mcl-1, STAT3 and p-STAT3 caused by fucoxanthin were inhibited. This is the first analysis of effects on SGC-7901 and BGC-823 cells by fucoxanthin. Fucoxanthin can induce cell-cycle arrest and apoptosis in these cells. These effects involved downregulation of Mcl-1, STAT3 and p-STAT3. This work is significant for better understanding of mechanisms leading to human gastric adenocarcinoma formation and informing exploitation of anti-tumor marine drug, and for providing Mcl-1 and STAT3 as potential therapeutic targets for gastric adenocarcinoma.     

Rhopaloic acid A induces apoptosis,autophagy and MAPK activation through ROS-mediated signaling in bladder cancer

BackgroundBladder cancer (BC) is a very common type of malignant cancer in men and new therapeutic strategies are urgently needed to reduce mortality. Several studies have demonstrated that Rhopaloic acid A (RA), a compound isolated from marine sponges, fights cancer but its potential anti-tumor effect on BC is still unknown.PurposeThe present study was aimed to explore the potential anti-tumor effects of RA against human BC cells and the underlying molecular mechanism.MethodsCell cytotoxicity was determined using the MTT and colony formation assays. Cell cycle distribution, apoptosis induction and generation of mitochondrial reactive oxygen species (ROS) were analyzed by flow cytometry. Mitochondrial membrane potential, acridine orange staining and intracellular ROS levels were observed using fluorescence microscopy. Levels of various signaling proteins were assessed using Western blotting. Furthermore, a zebrafish BC xenotransplantation model was used to confirm the anti-tumor effect of RA in vivo.ResultsTreatment with RA significantly suppressed the proliferation of BC cells that resulted from G2/M cycle arrest. Additionally, RA induced mitochondrial-mediated apoptosis and autophagy in BC cells. The death of BC cells induced by RA was rescued by treatment with inhibitors of apoptosis (Z-VAD-FMA) or autophagy (3-MA). RA activated the MAPK pathway and increased the production of cellular and mitochondrial ROS. Treatment with the ROS scavenger N-acetyl cysteine, effectively reversed the induction of apoptosis, autophagy, JNK activation and DNA damage elicited by RA. Finally, RA significantly inhibited tumor growth in a zebrafish BC xenotransplantation model.ConclusionTaken together, our findings indicate that RA induces apoptosis and autophagy and activates the MAPK pathway through ROS-mediated signaling in human BC cells. This RA-induced pathway offers insights into the molecular mechanism of its antitumor effect and shows that RA is a promising candidate for the treatment of BC.     

Gomisin A ameliorates metastatic melanoma by inhibiting AMPK and ERK/JNK-mediated cell survival and metastatic phenotypes

BackgroundGomisin A (G.A), a lignan compound extracted from the fruits of Schisandra chinensis, is known to exert anti-tumor effects on hepatocarcinoma and colorectal cancer cells. Suppression of proliferation and metastatic abilities of cancer cells are some effective cancer treatment methods.PurposeThe objective of this study is to investigate the effects of G.A on metastatic melanoma, and the mechanism by which it affects metastatic melanoma.Study designThe anti-proliferative and anti-metastatic effects of G.A were observed in in vitro and in vivo.MethodsWST assay and flow cytometry were conducted to investigate the effect of G.A on proliferation, cell cycle arrest, and apoptosis in metastatic melanoma cell lines. Migration and invasion abilities of G.A-treated melanoma cells were observed by wound healing and invasion assays.ResultsG.A (25–100 μM) decreased the viability of melanoma cells by inducing cell cycle arrest and apoptosis. These anti-proliferative effects of G.A were found to be mediated by AMPK, ERK, and JNK activation. G.A (5–20 μM) decreased the migration and invasion of melanoma cells by suppressing epithelial-mesenchymal transition (EMT). Consequently, G.A (2–50 mg/kg) inhibited lung metastasis by suppressing EMT and inducing cell cycle arrest and apoptosis in melanoma cells.ConclusionThese results conclude that G.A has the potential to reduce metastatic melanoma through its anti-proliferative and anti-metastatic effects.     

Harmine induces apoptosis and inhibits tumor cell proliferation,migration and invasion through down-regulation of cyclooxygenase-2 expression in gastric cancer

Cyclooxygenase-2 (COX-2) plays an important role in the carcinogenesis and progression of gastric cancer. Harmine is reported as a promising drug candidate for cancer therapy; however, effects and action mechanism of harmine on the human gastric cancer cells remain unclear. This study evaluated the anti-tumor effects of harmine on human gastric cancer both in vitro and in vivo. The cell proliferation was determined using MTT colorimetric assay. Apoptosis was measured by DAPI staining and flow cytometry analysis. The wound healing and transwell invasion assays were performed to evaluate the effects of harmine on the migration and invasion of gastric cancer cells. The expression of COX-2, proliferating cell nuclear antigen (PCNA), Bcl-2, Bax and matrix metalloproteinase-2 (MMP-2) was detected by Western blot analysis. Our results showed that harmine significantly inhibited cellular proliferation, migration, invasion and induced apoptosis in vitro, as well as inhibited tumor growth in vivo. In addition, harmine significantly inhibited the expression of COX-2, PCNA, Bcl-2 and MMP-2 as well as increased Bax expression in gastric cancer cells. These results collectively indicate that harmine induces apoptosis and inhibits proliferation, migration and invasion of human gastric cancer cells, which may be mediated by down-regulation of COX-2 expression.     

Cell cycle arrest and induction of apoptosis by cajanin stilbene acid from Cajanus cajan in breast cancer cells

Background: The low abundant cajanin stilbene acid (CSA) from Pigeon Pea (Cajanus cajan) has been shown to kill estrogen receptor α positive cancer cells in vitro and in vivo. Downstream effects such as cell cycle and apoptosis-related mechanisms have not been analyzed yet.Material and methods: We analyzed the activity of CSA by means of flow cytometry (cell cycle distribution, mitochondrial membrane potential, MMP), confocal laser scanning microscopy (MMP), DNA fragmentation assay (apoptosis), Western blotting (Bax and Bcl-2 expression, caspase-3 activation) as well as mRNA microarray hybridization and Ingenuity pathway analysis.Results: CSA induced G2/M arrest and apoptosis in a concentration-dependent manner from 8.88 to 14.79 µM. The MMP broke down, Bax was upregulated, Bcl-2 downregulated and caspase-3 activated. Microarray profiling revealed that CSA affected BRCA-related DNA damage response and cell cycle-regulated chromosomal replication pathways.Conclusion: CSA inhibited breast cancer cells by DNA damage and cell cycle-related signaling pathways leading to cell cycle arrest and apoptosis.